Localization of Homology Between Actinobacillus actinomycetemcomitans Chromosomal and Extrachromosomal DNA. A previously reported plasmid, pVT745, isolated from A. actinomycetemcomitans (Aa) strain VT745, hybridized under stringent conditions to genomic DNA from 15 Aa strain-specific patterns of hybridization with strains in this particular collection. Recently, pVT745 also hybridized to 9 of 15 Aa clinical isolates, revealing 3 additional patterns of hybridization. Since the hybridization patterns observed with genomic DNA clearly indicated that the intact plasmid was no inserting into the genomes of any of these 24 strains, the purpose of the current study was to characterize the plasmid and to locate in this molecule sequences responsible for hybridization to Aa genomic DNA. Construction of a restriction endonuclease map has revealed the size of this molecule to be approximately 25.1 Kb. pVT745 was cloned into 3 non- overlapping fragments (6.8, 8.0 and 10.3 Kb) that demonstrated no cross-hybridization by Southern blot analysis. However, genomic DNA representing each of the 5 strain-specific patterns hybridized with 1, 2, or 3 of the cloned sequences, suggesting that 2 or more unique sequences within pVT745 may be present in Aa genomic DNA. We hypothesize that these sequences may represent insertion sequence (IS) elements, transposons, or both. Current efforts are directed towards localization of regions of pVT745 responsible for hybridization to genomic DNA, and towards further characterization of the entire plasmid molecule.